Effect Of PBEF Expression On The Sodium Water Transport System Of Lung Epithelial Cells

Objective:Increased alveolar capillary permeability and decreased alveolar fluid clearance are the main causes of pulmonary edema after acute lung injury(ALI)after cardiopulmonary bypass.The sodium water transport system is the main motive force for the clearance of liquid in the alveolus.Our previous study found that the pre B cell clone enhancement factor(PBEF)is closely related to the increased permeability of the lung endothelial cells after cardiopulmonary bypass,and whether PBEF inhibits the clearance of the alveolar fluid in the pulmonary epithelial cells is still to be discussed.Therefore,this experiment is to explore the effect of PBEF expression on the sodium and water transport system of lung epithelial cells.Methods:1.RLE-6TN and R3/1 cells were isolated by primary generation.2.Immunofluorescence was used to identify the isolated cells as the target cells.3.Stable cell lines were constructed by transfection of pCAGGS PBEF plasmid and pCAGGS empty plasmid.4.A hypoxic reoxygenation model was constructed,which were divided into eight groups: RLE-6TN cell control group,RLE-6TN cell model group,RLE-6TN cell model-pCAGGS PBEF plasmid transfection group and RLE-6TN cell model-pCAGGS empty plasmid group.R3/1 cell control group,R3/1 cell model group,R3/1cell model,-pCAGGS PBEF plasmid transfection group,R3/1 cell model-pCAGGS empty plasmid group.5.Finally,we used ATP enzyme to detect NKA activity,and detected the expression of PBEF,ENaC,RT140 and surfactant protein(SP)by PCR and Western blot.Results:1.Fluorescence quantitative PCR detection: compared with the control group,after transfection of PBEF plasmid,the expression of two indexes of PBEF andRTI40 in RLE-6TN and R3/1 cells increased,while the expression of ENaC and SP decreased,and the results were statistically different(P<0.05).2.Western Blot detection: compared with the control group,after transfection of PBEF plasmid,the protein expression of PBEF and RTI40 in RLE-6TN and R3/1cells increased and the expression of ENaC and SP decreased,and the results were statistically different(P<0.05).3.ATP enzyme activity detection: compared with the control group,the expression of NKA was decreased in the transfected PBEF plasmid group and in RLE-6TN and R3/1 cells,and the results were statistically different(P<0.05).Conclusions:The expression of ENaC and SP increased after transfection of PBEF expression plasmid.It is indicated that the expression of major transport factors of sodium water transport system is inhibited,and the clearance function of fluid in alveolar fluid is also affected by the up regulation of PBEF.The decrease of SP expression indicates that the protective effect of alveolar epithelial cells is reduced,and the lung epithelial cells are more vulnerable to injury when stimulated by external stimuli.At the same time,ATP enzyme activity detection results confirmed that compared with the control group,the expression of NKA in the PBEF group decreased,and further proved that the expression of PBEF was up-regulated,which affected the clearance of pulmonary edema fluid.